Merocyanine 540 is a fluorescent probe which binds specifically to leukemia cell, but not mature blood cell, surfaces. During induced erythroleukemia differentiation in vitro, dye binding regions become localized to one site on the membrane, along with a subset of the major lectin receptors of the cell. Similar rearrangements are demonstrable in normal enucleating mouse erythroid cells, suggesting that differentiating erythroleukemia cells are preparing their cell surfaces for enucleation. Based on the greater affinity of MC540 for liquid phase phospholipid vesicles than for solid phase vesicles, we have suggested that these differentiation-related membrane events may involve differential association of surface receptors with regions of the plasma membrane of different phase state. This association keys a subset of surface molecules for elimination by the differentiating cell. We will test and extend this hypothesis by investigations of the relationship between MC540 staining and lipid composition, phase state and symmetry in normal plasma membranes and membranes whose lipid composition has been altered by insertion of liposomes or free fatty acids. We also propose to quantitate the phase state dependence of MC540 binding and its physical parameters, and to characterize various structural analogues for their usefulness in the design of photoaffinity labels or stains of altered toxicity. The relevance of MC540 binding regions to cell differentiation, physiology, and transformation will be investigated by examining inducible mouse and human myelocytic leukemia cell lines for their ability to bind MC540, and for the effects of cellular differentiation on the distribution of binding regions. Non-binding variants of stainable cells will be selected and examined for changes in their physiological and developmental characteristics. And finally, leukemia cells temperature-sensitive for maintenance of transformation will be studied to elucidate the relationship between MC540 binding and the transformed state.